Anandamide (arachidonyiethanolamide) is an endogenous compound that binds to one of the most ubiquitous receptors in the brain, the delta9- tetrahydrocannabinol (THC) receptor. Anandamide has been demonstrated to be a cannabinoid agonist and may serve as a genuine neurotransmitter. The long term goal of this project is to characterize the enzymes that are responsible for the metabolism of anandamide. From the properties of the enzymes, it will be possible to design a series of inhibitors to prevent the breakdown of anandamide. The development of drugs that block anandamide's breakdown will be important for therapeutics since THC receptors mediate behavior involving memory, appetite, movement, pain, and mood. Preliminary studies indicate that enzymatic activities in the brain, do indeed, mediate the formation of anandamide from arachidonic acid and ethanolamine (a synthase) and the breakdown of anandamide to arachidonic acid (an amidase). The specific goals of this proposal are the following: 1) to characterize these enzymes in terms of kinetics and substrate specificity. Kinetic experiments will be conducted to determine the effects of time, temperature, pH, ions, substrate and enzyme concentrations upon the rate of reaction. In order to determine enzyme specificity for the amidase and the synthase reactions, a series of: fatty acid ethanolamide analogs of arachidonylethanolamide and a series of fatty acids analogs of arachidonic acid, respectively, will be tested as competitive inhibitors and/or substrates analogs for these enzymatic reactions; 2) to synthesize a series of ketonic arachidonylethanolamide analog "transition" state inhibitors, such as trifluoromethyl ketones, alpha-keto ester derivatives, or alpha-keto amide derivatives. The ability of these compounds to inhibit the amidase in tissue preparations and cell culture will be determined. In addition, these inhibitors will be tested for their effect upon the synthase, their ability to bind to the THC receptor, and their ability to potentiate the effect of anandamide in the rat vas deferens twitch response assay. Preliminary studies indicate that one of these compounds (an alpha-keto ester) is a potent inhibitor of anandamide amidase with a Ki in the submicromolar range and that it does not bind tightly to the THC receptor. The inhibitors discovered in these studies could be radiolabelled and used as probes for the detection of the amidase in brain slices; 3) to determine the subcellular localization of these enzymatic activities in the nerve terminals and to purify the enzymes. The enzymes will be purified by a combination of classical and affinity chromatography procedures. The purified enzymes will be utilized for the enzymatic studies described above, and for partial amino acid sequence determination. The purified protein could be used for the production of antibodies for immunocytochemical localization of the enzymes involved in anandamide metabolism.